Original research
Effect of bromhexine in hospitalized patients
For numbered affiliations see
end of article.
Correspondence to
Dr Farzaneh Dastan,
Department of Clinical
Pharmacy, School of
Pharmacy, Shahid Beheshti
University of Medical
Sciences, Tehran, Iran;
​fzh.​dastan@g​ mail.​com
Accepted 24 February 2021
© American Federation for
Medical Research 2021.
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rights and permissions.
Published by BMJ.
To cite: Tolouian R,
Mulla ZD, Jamaati H,
et al. J Investig Med Epub
ahead of print: [please
include Day Month Year].
doi:10.1136/jim-2020-
001747
with COVID-19
Ramin Tolouian ‍ ‍,1 Zuber D Mulla ‍ ‍,2 Hamidreza Jamaati,3
Abdolreza Babamahmoodi,4 Majid Marjani,5 Raha Eskandari,3 Farzaneh Dastan6
ABSTRACT
Background Bromhexine is a potent inhibitor
of transmembrane serine protease 2 and appears
to have an antiviral effect in controlling influenza
and parainfluenza infection; however, its efficacy in
COVID-19 is controversial.
Methods A group of hospitalized patients with
confirmed COVID-19 pneumonia were randomized
using 1:1 allocation to either standard treatment
lopinavir/ritonavir and interferon beta-­1a or
bromhexine 8 mg four times a day in addition to
standard therapy. The primary outcome was clinical
improvement within 28 days, and the secondary
outcome measures were time to hospital discharge,
all-­cause mortality, duration of mechanical
ventilation, the temporal trend in 2019-­nCoV reverse
transcription-­polymerase chain reaction positivity
and the frequency of adverse drug events within 28
days from the start of medication.
Results A total of 111 patients were enrolled in
this randomized clinical trial and data from 100
patients (48 patients in the treatment arm and 52
patients in the control arm) were analyzed. There
was no significant difference in the primary outcome
of this study, which was clinical improvement. There
was no significant difference in the average time
to hospital discharge between the two arms. There
were also no differences observed in the mean
intensive care unit stay, frequency of intermittent
mandatory ventilation, duration of supplemental
oxygenation or risk of death by day 28 noted
between the two arms.
Conclusion Bromhexine is not an effective
treatment for hospitalized patients with COVID-19.
The potential prevention benefits of bromhexine
in asymptomatic postexposure or with mild
infection managed in the community remain to be
determined.
INTRODUCTION
The COVID-19 pandemic remains one of the
major public health issues, despite preven-
tive measures such as wearing mask and social
distancing, being implemented worldwide.1
The search for finding the effective treatment
to prevent or treat the viral infection is ongoing
but, so far, has had limited success.
Bromhexine is an inexpensive and widely
available medication with a low side-­ effect
profile and has been used as mucolytic in
Tolouian R, et al. J Investig Med 2021;0:1–6. doi:10.1136/jim-2020-001747
J Investig Med: first published as 10.1136/jim-2020-001747 on 15 March 2021. Downloaded from http://jim.bmj.com/ on August 11, 2021 by guest. Protected by copyright.
Significance of this study
What is already known about this subject?
►► The COVID-19 pandemic remains one of
the major public health issues, despite
preventive measures being implemented
worldwide.
►► Bromhexine is a potent inhibitor of
transmembrane serine protease 2 and has
an antiviral effect.
►► One study has shown the clinical benefit of
this inexpensive medicine in patients with
COVID-19 pneumonia.
What are the new findings?
►► Bromhexine is not an effective treatment in
hospitalized patients with COVID-19.
►► Presence of renal disease is the strongest
predictor of mortality in hospitalized
patients with COVID-19 in our multivariate
analysis.
►► Bromhexine as a transmembrane serine
protease 2 inhibitor could not reduce
duration of hospitalization.
►► Bromhexine as a transmembrane serine
protease 2 inhibitor could not reduce the
need for mechanical ventilation compared
to the control.
How might these results change the focus
of research or clinical practice?
►► The potential prevention benefits of
bromhexine in asymptomatic postexposure
patients or those with mild infection
managed out of medical centers remain to
be determined.
different respiratory conditions since 1963.2
Bromhexine is a potent inhibitor of transmem-
brane serine protease 2 (TMPRSS2) and seems
to have an antiviral effect. It has been shown
that the presence of TMPRSS2 is very essential
for influenza virus infection and propagation.
Bromhexine has been shown to be effective in
controlling influenza infection by blocking the
cleavage of the surface glycoprotein hemagglu-
tinin of the influenza virus.3 4
Researchers have proposed that bromhexine
may be an effective option to reduce primary
transmission, viral load, dissemination and
1
 Original research
secondary replication of SARS-­ CoV-2.5–8 COVID-19, like
SARS-­CoV, binds to human ACE 2 via its spike glycoprotein
(S-­protein) expressed on its envelope for entering the target
cell. S protein is composed of one amino-­terminal (S1) and
one carboxy-­terminal (S2). Cleavage at the S1–S2 junction
by protease (TMPRSS2) is essential to prime the virus spikes
and activate membrane fusion. It has also been proposed that
bromhexine, by blocking non-­endosomal pathways via serine
protease 2 (TMPRSS2), theoretically blocks the priming of
the spikes and virus entry into the host cell.9 10
In a small open-­label trial, the clinical benefit of brom-
hexine administration in patients with SARS-­CoV-2 pneu-
monia has been reported.11 This study tested whether
bromhexine hydrochloride was an effective medication to
improve clinical outcomes and mortality in hospitalized
patients with COVID-19.
Materials and methods
This clinical trial was designed as a randomized, single
center, open-­ label study. From 156 patients who were
screened, in Masih Daneshvari Hospital, a tertiary and
referral center for COVID-19, 111 patients with a diagnosis
of COVID-19 pneumonia were enrolled. The study began
on May 6, 2020, and enrollment of patients was completed
on June 20, 2020. Written informed consent from all the
study subjects was obtained.
Patients were randomized at a 1:1 ratio to receive either
oral bromhexine in addition to standard therapy or stan-
dard therapy alone. Subjects who were enrolled received
a trial number. Every single trial number was randomized
to either arm of the study through computer randomiza-
tion. The study was randomized, controlled, and open-­
labeled, and the trial was monitored by the data monitoring
committee. Trial recruitment stopped after the target study
population had been reached and was closed when all of the
patients had completed their follow-­up visit.
Inclusion criteria
The inclusion criteria were as follows: hospital admission,
18 years old or greater at the time of signing the informed
consent, chest imaging and clinical symptoms consistent
with COVID-19 pneumonia, laboratory (reverse transcrip-
tion polymerase chain reaction (RT-­PCR)) confirmed infec-
tion with 2019-­nCoV, willingness to participate in the study,
and no concurrent participation in other clinical trials.
Exclusion criteria
The following exclusion criteria were used: pregnancy or
lactation, severe liver disease (eg, aspartate aminotrans-
ferase (AST)>5 times upper limit), undergoing dialysis
or transferred to another hospital within 72 hours and a
history of allergy to bromhexine.
Standard arm
Patients received treatment based on the hospital COVID-19
treatment protocol and best practice guidelines in place at
that time. (lopinavir/ritonavir) (Kaletra) 400/100 two times
per day for 7 days or discharge from hospital and interferon
(IFN) beta-­1a (Rebif) 44 μg subcutaneous every other day
for five doses in addition to supportive and symptomatic
therapy.
2 Tolouian R, et al. J Investig Med 2021;0:1–6. doi:10.1136/jim-2020-001747
Treatment arm
J Investig Med: first published as 10.1136/jim-2020-001747 on 15 March 2021. Downloaded from http://jim.bmj.com/ on August 11, 2021 by guest. Protected by copyright.
The treatment arm received oral bromhexine hydrochloride
8 mg four times a day for 2 weeks in addition of standard
therapy.
Outcome measures
The primary outcome was clinical improvement within 28
days. Clinical improvement was defined as the time (in days)
from initiation of the study treatment (active or placebo) until
a decline of two categories on a clinical status scale occurred.
The six-­category ordinal scale of clinical status which ranged
from hospital discharge to death and is itemized as follows:
(1) hospital discharge or meeting discharge criteria (discharge
criteria are defined as clinical recovery, ie, fever, respiratory
rate, oxygen saturation returning to normal, and cough
relief); (2) non-­intensive care unit (ICU) hospitalization, not
requiring supplemental oxygen; (3) non-­ICU hospitalization,
requiring supplemental oxygen (but not noninvasive venti-
lation/high-­flow nasal cannula); (4) ICU/non-­ICU hospital-
ization, requiring noninvasive ventilation/high-­ flow nasal
cannula therapy; (5) ICU hospitalization, requiring invasive
mechanical ventilation; and (6) death.
The criteria for ICU admission were worsening of respira-
tory distress assessed by the physician, hemodynamic insta-
bility requiring vasopressors, and oxygen desaturation of
<85% that was not responsive to low-­flow oxygen therapy.
Secondary outcome measures included time to hospital
discharge, all-­ cause mortality, duration of mechanical
ventilation, time to 2019-­ nCoV RT-­ PCR negativity and
frequency of serious adverse drug events, within 28 days
from the start of medication.
Statistical analysis
Data were analyzed using SAS V.9.4. The distribution of
the demographic and clinical characteristics of the sample
was summarized by treatment status. Number and percent
were reported for binary outcomes. Means and SD were
calculated for continuous outcomes such as time to hospital
discharge. Associations between the treatment status and
patient characteristics were tested for statistical significance
using χ2 or Fisher’s exact test, as appropriate, for categor-
ical variables, and two-­sample t-­tests for continuous vari-
ables. An alpha of 0.05 was used for all significance testing.
Study subjects were tested for COVID-19 using a poly-
merase chain reaction (PCR) test on days 1, 7, and 28. The
prevalence of PCR test positivity was plotted by time. A
longitudinal data analysis using generalized estimating equa-
tions was attempted. However, there was an error in the
estimation routine when fitting the generalized estimating
equations logistic regression model and the convergence
was questionable. Standard errors could not be generated.
Kaplan-­Meier curves were created for time to improvement.
Wilcoxon tests (rather than log-­rank tests) were performed
to determine if the survival curves for the treatment groups
differed from one another in the population. The assumption
of proportional hazards was violated for multiple predic-
tors for both outcomes, time to improvement and time to
death. Given these violations, HRs from Cox (proportional
hazards) regression models were not calculated. Instead, ORs
for improvement and mortality were calculated from logistic
regression models and reported with 95% CIs and p values.
 Original research

J Investig Med: first published as 10.1136/jim-2020-001747 on 15 March 2021. Downloaded from http://jim.bmj.com/ on August 11, 2021 by guest. Protected by copyright.
Table 1 Demographic and clinical characteristics of the 100
study subjects*
Bromhexine Placebo
Characteristics (n=48) (n=52) P value
Age (years), mean (SD) 50.7 (16.4) 53.1 (15.2) 0.44
Male, n (%) 22 (45.8) 24 (46.2) 0.97
Married 40 (83.3) 46 (88.5) 0.46
BMI, mean (SD) 26.2 (1.8) 33.2 (4.5) <0.0001
Obese (BMI≥30 kg/m2), n (%) 0 (0.0) 47 (90.4) <0.0001
Smoker, n (%) 4 (8.3) 9 (17.3) 0.18
Traveled, n (%) 7 (14.6) 10 (19.2) 0.54
Exposure to a COVID-19 case 8 (16.7) 16 (30.8) 0.10
prior to infection, n (%)
Blood group, n (%) 0.91
 A+ 10 (20.8) 11 (21.2)
 A− 1 (2.1) 4 (7.7)
 B+ 11 (22.9) 8 (15.4)
 B− 2 (4.2) 2 (3.9)
 O+ 16 (33.3) 18 (34.6)
 O− 1 (2.1) 2 (3.9)
 AB+ 4 (8.3) 3 (5.8)
Figure 1 CONSORT flow diagram. Patient enrollment and  AB− 3 (6.3) 4 (7.7)
treatment assignment. Comorbidities, n (%)
 Asthma 3 (6.3) 3 (5.8) 1.0
Given the imbalanced distribution of several factors of clinical  Autoimmune disease 4 (8.3) 1 (1.9) 0.19
significance between the two study arms, ORs were adjusted  Cancer 3 (6.3) 3 (5.8) 1.0
for obesity (defined as a Body Mass Index (BMI) of ≥30 kg/  Cerebrovascular accident 1 (2.1) 1 (1.9) 1.0
m2), smoking, and renal disease (defined as an estimated  Chronic obstructive 3 (6.3) 4 (7.7) 1.0
glomerular filtration rate between 16 and 60 mL/min). Sparse pulmonary disease
data bias was a possibility, given the small number of patients  Coronary heart disease 6 (12.5) 3 (5.8) 0.31
who did not improve and the small number of deaths. To  Diabetes 16 (33.3) 17 (32.7) 0.95
minimize the risk of triggering sparse data bias, Firth’s penal-  Hypertension 20 (41.7) 19 (36.5) 0.60
ized maximum likelihood estimation was used when esti-  Liver diagnosis 2 (4.2) 1 (1.9) 0.61
mating the unadjusted and adjusted ORs for both death and  Renal diagnosis 4 (8.3) 1 (1.9) 0.19
improvement.12
*Data are reported as mean and SD for continuous variables and number (n)
and percent for categorical variables.
RESULTS BMI, Body Mass Index.
A total of 156 patients with proven COVID-19 pneu-
monia were screened. Forty-­five of them were excluded
(33 patients were enrolled in another experimental trial, 7 such as asthma, hypertension, diabetes, chronic obstructive
were on hemodialysis, 3 had severe liver disease and 2 were pulmonary disease, cancer and cerebrovascular accident
transferred to another hospital). A total of 111 patients were almost identical between the study arms.
were enrolled in this randomized clinical trial. They were
assigned to either the treatment with bromhexine group or
the standard treatment group in a 1:1 ratio with 59 patients Primary clinical outcome
in the treatment arm and 52 patients in the standard/control There was no significant difference in the primary outcome
arm. Eleven patients were lost to follow-­up in the treatment of this study, which was time to clinical improvement. The
arm. No attrition occurred in the control arm. Data from median time to improvement in the bromhexine arm was
the total of 100 patients (48 patients in the treatment arm 7 days, while that in the control arm it was 6 days. The p
and 52 patients in the control arm) were analyzed (figure 1). value from the Wilcoxon test for equality of the survival
The distributions of most of the demographic and disease curves in the population was 0.61 (figure 2).
characteristics were similar in the treatment and standard The unadjusted OR for clinical improvement comparing
groups (table 1). patients in the bromhexine arm with those in the stan-
The mean age±SD was 50.7±16.4 years among the dard treatment arm was 0.92. After adjusting for obesity,
treated arm and 53.1±15.2 in the standard arm. In terms smoking, and renal disease, this OR was 4.15 (95% CI 0.13
of gender, the percentage of men in both the treatment to 138.25, p=0.43) (table 2).
and standard groups was approximately 46%. There was a Patients with no renal disease had 25 times the odds
significant difference (p<0.0001) in the mean BMI between of improving compared to patients with renal disease
the treatment group (26.2±1.8) and the standard treatment (1/0.04=25): adjusted OR=0.04, 95% CI 0.004 to 0.42,
group (33.2±4.5). The distribution of other comorbidities p=0.007.
Tolouian R, et al. J Investig Med 2021;0:1–6. doi:10.1136/jim-2020-001747 3
 Original research
Figure 2 Kaplan-­Meier estimates of cumulative clinical
improvement. The median time to clinical improvement was
7.0 days (95% CI 6.0 to 7.0) in the bromhexine arm and 6.0 days
(95% CI 6.0 to 7.0) in the placebo arm.
Unadjusted and adjusted ORs for death are listed in
table 3.
The unadjusted OR for death comparing patients in the
bromhexine group with those in the control arm was 1.09.
After adjustment for obesity, smoking, and renal disease, the
bromhexine OR was 0.24. Given this result, it appears, at
first, that there was a 76% reduction in the odds of dying
(bromhexine vs standard treatment); however, this result was
not statistically significant: 95% CI:0.007 to 8.03, p=0.43. In
a similar fashion, the obesity OR was also affected by strong
joint confounding by the remaining three variables found
in the multiple logistic regression model. The confounding
was severe enough to reverse the direction of the associa-
tion: unadjusted obesity, OR=1.13, and adjusted obesity,
OR=0.48. Neither of the obesity ORs were statistically signif-
icant. In contrast, both the unadjusted and adjusted ORs for
mortality for the presence of renal disease were above 1 and
statistically significant: adjusted renal disease, OR=24.98,
95% CI 2.40 to 259.71, p=0.007.
Secondary clinical outcomes
There was no significant difference in the mean (average)
time to hospital discharge between the two arms. There
were also no differences observed in the mean ICU stay,
frequency of intermittent mandatory ventilation, duration
Table 2 Unadjusted and adjusted ORs* for clinical improvement in 100 patients with COVID-19: 96 improved vs 4 did not improve
Unadjusted
Risk factor (sample size) OR
Bromhexine (n=48) vs standard treatment (n=52) 0.92
Obese‡ (n=47) vs not obese (n=53) 0.88
Renal disease (n=5) vs no renal disease (n=95) 0.04
Smoker (n=13) vs non-­smoker (n=87) 1.46
*ORs were calculated from logistic regression models that used Firth’s penalized maximum likelihood estimation.
†Each OR is adjusted for the remaining variables that are found in the table.
‡Defined as a Body Mass Index of ≥30 kg/m2.
4 Tolouian R, et al. J Investig Med 2021;0:1–6. doi:10.1136/jim-2020-001747
of supplemental oxygenation and risk of death by day 28
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noted between the two arms. The prevalence of the use of
high-­flow nasal oxygenation in the bromhexine group was
significantly higher than the prevalence in the standard arm
(56.3% vs 23.1%, p=0.001) (table 4).
The temporal trend in the probability of being PCR posi-
tive was assessed. On days 1, 7, and 28, 100%, 60.4%, and
0%, respectively, of the patients in the bromhexine arm
were PCR positive. Among patients in the standard arm,
the prevalence of PCR positivity on days 1, 7, and 28 were
100%, 34.6%, and 0%, respectively.
Adverse events
No major adverse events were noted.
DISCUSSION
This open-­label, randomized, single-­center, controlled trial
determined that bromhexine was not an effective treatment
in hospitalized patients with COVID-19. Our data do not
show a benefit of bromhexine in regard to clinical improve-
ment, ICU admissions, the need for mechanical ventilation,
or all-­cause mortality.
The goal of randomization is to balance the distribution
of known and unknown confounders between the two arms
of the study, thereby reducing the possibility of confounding
by these factors. At times, the desired balancing is not
achieved with random allocation. In our study, the patients
in the standard arm had a higher prevalence of obesity
(90.4%) compared with those in the bromhexine arm
(0.0%): p<0.0001.
It has been observed that obese patients have higher
mortality in COVID-19 infection than non-­ obese
patients.13 14 Clinicians, no doubt, would like to know if
the effect of bromhexine on the risk of mortality varies by
obesity status. Is it possible that among obese patients, treat-
ment with bromhexine has a different effect on mortality
than in patients who are not obese? We are unable to answer
this question via a stratified analysis, given that none of our
bromhexine patients were obese. Future similar studies
should be powered in such a fashion as to be able to assess
if there is an interaction between bromhexine and obesity
when mortality is the outcome.
Regarding the mortality, our data showed that the pres-
ence of renal disease was strongly correlated in a positive
fashion with the outcome of death in both the unadjusted
and adjusted analyses (table 3). The adjusted OR for clinical
improvement comparing patients with renal disease to those
without renal disease is 0.04. ORs possess the reciprocal
Adjusted†
P
95% CI P value OR 95% CI value
0.15 to 5.66 0.93 4.15 0.13 to 138.25 0.43
0.14 to 5.43 0.89 2.11 0.08 to 56.39 0.66
0.004 to 0.33 0.003 0.04 0.004 to 0.42 0.007
0.07 to 31.67 0.81 0.99 0.05 to 18.14 0.99
 Original research

J Investig Med: first published as 10.1136/jim-2020-001747 on 15 March 2021. Downloaded from http://jim.bmj.com/ on August 11, 2021 by guest. Protected by copyright.
Table 3 Unadjusted and adjusted ORs* for time to death in 100 patients with COVID-19: 4 died vs 96 survived
Unadjusted Adjusted†
P
Risk factor (sample size) OR 95% CI P value OR 95% CI value
Bromhexine (n=48) vs standard treatment (n=52) 1.09 0.18 to 6.67 0.93 0.24 0.007 to 8.03 0.43
Obese‡ (n=47) vs not obese (n=53) 1.13 0.18 to 6.96 0.89 0.48 0.02 to 12.71 0.66
Renal disease (n=5) vs no renal disease (n=95) 26.72 3.02 to 236.50 0.003 24.98 2.40 to 259.71 0.007
Smoker (n=13) vs non-­smoker (n=87) 0.69 0.03 to 14.96 0.81 1.02 0.06 to 18.67 0.99
*ORs were calculated from logistic regression models that used Firth’s penalized maximum likelihood estimation.
†Each OR is adjusted for the remaining variables that are found in the table.
‡Defined as a Body Mass Index of ≥30 kg/m2.

property, and therefore the adjusted OR for clinical improve- between our study and theirs, such as being a single-­center
ment comparing patients free of renal disease to those who and open-­label trial, there are some obvious differences. A
have renal disease is 1/0.04 or 25. This adjusted OR of 25 higher number of patients were enrolled in our study (100
is statistically significant (p=0.007) and indicates the pres- vs 78 patients) and our data did not support the effective-
ence of a very strong relationship between this risk factor and ness of bromhexine in hospitalized patients with COVID-19.
the outcome of clinical improvement. Similarly, the adjusted One of the other differences between these two similar trials
OR for mortality for the presence of renal disease is approxi- was the standard therapy regimen. Our standard treatment
mately 25 and statistically significant. Our finding is in agree- was the combination of lopinavir/ritonavir (Kaletra) and IFN
ment with other reports that indicated renal disease is a strong beta-­1a (Rebif), while in the other study, the majority were
predictor of mortality in COVID-19 infection.15 16 taking hydroxychloroquine (HCQ). In the RECOVERY trial,
It has also been reported that people with the blood Kaletra was found to be ineffective in hospitalized patients
group of O and Rh− have lower risk of COVID-19 infec- with COVID-19.18 Additionally, the results from the Soli-
tion.17 None of the four decedents in our study were Rh− darity Trial were disappointing and did not show any benefit
but three of them were O positive. of subcutaneous IFN beta-1 (Rebif) in the same population.19
The most common use of bromhexine is as a mucolytic It is plausible that the combination of HCQ and bromhexine
cough suppressant in respiratory diseases. Bromhexine has is the key to improvement, but studies need to evaluate and
no Food and Drug Administration approval in the USA but assess this hypothesis.20 There is one ongoing C​ linicalTrials.​
has been used all over the world for more than 50 years.2 gov Identifier: NCT04355026 that may find the answer to
This inexpensive medication that blocks (TMPRSS2) might this question.
interfere with the process of cell entry of the SARS-­CoV-2.10 Our data also showed a higher proportion of patients
A small, single-­center, open-­label study showed that brom-
needing high-­ flow oxygenation support. The interaction
hexine is an effective medication that reduced the rate of ICU
of ACE2 receptor and TMPRSS2 with the S-­ protein in
admission, need for mechanical ventilation and mortality
SARS-­CoV-2 was studied by Hörnich et al. They claimed
in patients who suffer from SARS-­ CoV-2 pneumonia,
that SARS-­CoV-2 does not require TMPRSS2 on target cells
and concluded that bromhexine treatment may result in a
for cell–cell fusion and suggested that bromhexine is inef-
milder course of the disease.11 Despite the many similarities
fective in COVID-19 infection. They also moved one step
further and claimed that bromhexine may even moderately
enhance fusion. Although their paper has yet to be peer
Table 4 Outcomes of the 100 study subjects
reviewed, they concluded that SARS-­CoV-2 fusion with the
Bromhexine Placebo host cell, as compared with the first SARS virus, depends
Characteristic (n=48) (n=52) P value
more on the expression of the ACE2 receptor than protease
Time to hospital discharge, 9.1 (3.1) 9.2 (3.4) 0.82 activation.21 Possible enhancement of fusion by bromhexine
mean (SD)
may explain why patients in our Bromhexine group were
ICU admission (days), mean (SD) 0.6 (2.1) 0.8 (2.0) 0.67 more than twice as likely to have received high-­flow oxygen
No ICU admission and no 0 (0.0) 0 (0.0) – therapy than patients in our standard treatment arm.
oxygen, n (%)
The other counterintuitive finding in our study was the
No ICU admission and yes 43 (89.6) 47 (90.4) 1.0
higher percentage of PCR test positivity on day 7 in the
oxygen, n (%)
bromhexine arm (60.4%) compared with the standard arm
High-­flow nasal cannula oxygen, 27 (56.3) 12 (23.1) 0.001
n (%) (34.6%). While the prevalence of a positive PCR test was
Intermittent mandatory 5 (10.4) 5 (9.6) 1.0
75% higher in the bromhexine group than in the standard
ventilation, n (%) treatment group on day 7, we do not advise scrutinizing
Duration of supplemental 6.8 (2.2) 7.0 (3.3) 0.71 the results at a single point in time. A proper longitudinal
oxygen, mean (SD) data analysis would incorporate information from all of
Deaths by day 28, n (%) 2 (4.2) 2 (3.9) 1.0 the time points. We attempted to conduct such an analysis
*Data are reported as mean and SD for continuous variables and number (n) using generalized estimating equations logistic regression;
and percent for categorical variables. however, an error in the estimation routine was encoun-
ICU, intensive care unit. tered while fitting this model. This error was most likely
Tolouian R, et al. J Investig Med 2021;0:1–6. doi:10.1136/jim-2020-001747 5
 Original research
due to the fact that there was insufficient variation in the
prevalence of test positivity. Measurement of the outcome
at additional time points was most likely required.
Our trial had some limitations. The trial was a single-­
center, single-­country investigation, and hence general-
izability may be reduced. Additionally, our study was not
blinded. One of the purposes of blinding the study inves-
tigators to the treatment allocation is to reduce the chance
that the outcomes were assessed differently between the two
study arms.22 However, one of our endpoints was all-­cause
mortality, an objective rather than a subjective outcome.
Finally, given the small number of patients who did not
improve and the small number of patients who died, there
was a strong possibility that our ORs would be affected by
sparse data bias. To reduce the chance of sparse data bias,
Firth’s penalized likelihood method was used.12
CONCLUSION
Our findings indicate that bromhexine is not an effective
treatment for hospitalized patients with COVID-19, and
renal disease was the strongest predictor of mortality in our
multivariate analysis. Our study did not address bromhex-
ine’s use as prophylaxis. The potential prevention benefits
of bromhexine in asymptomatic postexposure patients or
those with mild infection managed out of medical centers
remain to be determined.
Author affiliations
1
Renal Section, Southern Arizona VA Health Care System, University of Arizona,
Tucson, Arizona, USA
2
Department of Obstetrics and Gynecology, and Office of Faculty Development,
Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center
El Paso, El Paso, Texas, USA
3
Chronic Respiratory Diseases Research Center (CRDRC), National Research
Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti
University of Medical Sciences, Tehran, Iran
4
Infectious Diseases and Tropical Medicine Research Center, Shahid Beheshti
University of Medical Sciences, Tehran, Iran
5 12 Fernandez NP, Mulla ZD. Avoiding sparse data bias: an example from
Clinical Tuberculosis and Epidemiology Research Center, National Research
Institute of Tuberculosis and Lung Diseases (NRITLD), Masih Daneshvari
Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran
6 diagnosed with COVID-19: results from an integrated health care organization.
Department of Clinical Pharmacy, School of Pharmacy, Shahid Beheshti
University of Medical Sciences, Tehran, Iran
Acknowledgements The authors thank Dr M B Shadmehr and Ms F
Ghorbani for organizing the team and facilitating the process.
Contributors RT, ZDM, and FD were involved in design, analysis of data and
drafting of the manuscript. HJ, AB, MM and RE were involved in recruiting
patients, management and follow up. Data collection was done by AB and FD.
All authors read and signed the final paper.
Funding The authors have not declared a specific grant for this research from
any funding agency in the public, commercial or not-­for-­profit sectors.
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval The study was approved by the ethics committee of the
Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.NRITLD.
REC.1399.142).
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in
the article or uploaded as supplementary information. The deidentified subject
data are available in a secure space under the control of corresponding author
[​fzh.​dastan@g​ mail.​com].
6 Tolouian R, et al. J Investig Med 2021;0:1–6. doi:10.1136/jim-2020-001747
This article is made freely available for use in accordance with BMJ’s website
J Investig Med: first published as 10.1136/jim-2020-001747 on 15 March 2021. Downloaded from http://jim.bmj.com/ on August 11, 2021 by guest. Protected by copyright.
terms and conditions for the duration of the covid-19 pandemic or until
otherwise determined by BMJ. You may use, download and print the article for
any lawful, non-­commercial purpose (including text and data mining) provided
that all copyright notices and trade marks are retained.
ORCID iDs
Ramin Tolouian http://​orcid.​org/​0000-​0003-​2242-​9310
Zuber D Mulla http://​orcid.​org/​0000-​0003-​1670-​5702
REFERENCES
1 Hamidian Jahromi A, Mazloom S, Ballard DH. What the European and
American health care systems can learn from China COVID-19 epidemic; action
planning using purpose designed medical telecommunication, courier services,
home-­based quarantine, and COVID-19 walk-­in centers. Immunopathol Persa
2020;6:e17.
2 Zanasi A, Mazzolini M, Kantar A. A reappraisal of the mucoactive activity and
clinical efficacy of bromhexine. Multidiscip Respir Med 2017;12:7.
3 Böttcher E, Matrosovich T, Beyerle M, et al. Proteolytic activation of influenza
viruses by serine proteases TMPRSS2 and HAT from human airway epithelium. J
Virol 2006;80:9896–8.
4 Cheng Z, Zhou J, To KK-­W, et al. Identification of TMPRSS2 as a Susceptibility
Gene for Severe 2009 Pandemic A(H1N1) Influenza and A(H7N9) Influenza. J
Infect Dis 2015;212:1214–21.
5 Tolouian R, Tolouian AC, Ardalan M. Blocking serine protease (TMPRSS2) by
bromhexine; looking at potential treatment to prevent COVID-19 infection.
Marshall Journal of Medicine 2020;6:11.
6 Sagawa T, Inoue K-­I, Takano H. Use of protease inhibitors for the prevention of
COVID-19. Prev Med 2020;141:106280.
7 Barzegar A, Ghadipasha M, Rezaei N, et al. New hope for treatment
of respiratory involvement following COVID-19 by bromhexine. J
Nephropharmacol 2021;10:e11.
8 Depfenhart M, de Villiers D, Lemperle G, et al. Potential new treatment
strategies for COVID-19: is there a role for bromhexine as add-­on therapy?
Intern Emerg Med 2020;15:801–12.
9 Glowacka I, Bertram S, Müller MA, et al. Evidence that TMPRSS2 activates the
severe acute respiratory syndrome coronavirus spike protein for membrane
fusion and reduces viral control by the humoral immune response. J Virol
2011;85:4122–34.
10 Hoffmann M, Kleine-­Weber H, Schroeder S, et al. SARS-­CoV-2 cell entry
depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease
inhibitor. Cell 2020;181:271–80.
11 Ansarin K, Tolouian R, Ardalan M, et al. Effect of bromhexine on clinical
outcomes and mortality in COVID-19 patients: a randomized clinical trial.
BioImpacts 2020;10:209–15.
gynecologic oncology. J Registry Manag 2012;39:167–71.
13 Tartof SY, Qian L, Hong V, et al. Obesity and mortality among patients
Ann Intern Med. 2020;173:773–81. 11.
14 Chegini R, Bolurian A, Mojtahed Z, et al. High risk individuals in COVID-19
pandemic; an updated review. Immunopathol Persa 2021;7:e25.
15 Flythe JE, Assimon MM, Tugman MJ, et al. Characteristics and outcomes
of individuals with pre-­existing kidney disease and COVID-19 admitted to
intensive care units in the United States. Am J Kidney Dis 2021;77:190–203.
16 Gansevoort RT, Hilbrands LB. CKD is a key risk factor for COVID-19 mortality.
Nat Rev Nephrol 2020;16:705–6.
17 Ray JG, Schull MJ, Vermeulen MJ, et al. Association Between ABO and Rh Blood
Groups and SARS-­CoV-2 Infection or Severe COVID-19 Illness : A Population-­
Based Cohort Study. Ann Intern Med 2020.
18 Group RC. Lopinavir–ritonavir in patients admitted to hospital with COVID-19
(RECOVERY): a randomised, controlled, open-­label, platform trial. The Lancet
2020;396.
19 , Pan H, Peto R, et al, WHO Solidarity Trial Consortium. Repurposed Antiviral
Drugs for Covid-19 - Interim WHO Solidarity Trial Results. N Engl J Med.
2021;384:497–511. 02.
20 Tolouian R, Mulla Z. Controversy with bromhexine in COVID-19; where we
stand. Immunopathol Persa 2021;7:e12.
21 Hörnich B, Großkopf A, Schlagowski S, et al. SARS-­CoV-2 differs from SARS-­
CoV in the requirements for receptor expression and proteolytic activation to
trigger cell-­cell fusion and is not inhibited by bromhexine. July 2020.
22 Karanicolas PJ, Farrokhyar F, Bhandari M. Practical tips for surgical research:
blinding: who, what, when, why, how? Can J Surg 2010;53:345–8.